Herb pair of Huangqi-Danggui exerts anti-tumor immunity to breast cancer by upregulating PIK3R1

Abstract

Background: According to traditional Chinese medicine (TCM), drugs supplementing the vital energy, Qi, can eliminate tumors by restoring host immunity. The objective of this study is to investigate the underlying immune mechanisms of anti-tumor activity associated with Qi-supplementing herbs, specifically the paired use of Huangqi and Danggui.

Methods: Analysis of compatibility regularity was conducted to screen the combination of Qi-supplementing TCMs. Using the MTT assay and a transplanted tumor mice model, the anti-tumor effects of combination TCMs were investigated in vitro and in vivo. High content analysis and flow cytometry were then used to evaluate cellular immunity, followed by network pharmacology and molecular docking to dissect the significant active compounds and potential mechanisms. Finally, the anti-tumor activity and the mechanism of the active ingredients were verified by molecular experiments.

Results: There is an optimal combination of Huangqi and Danggui that, administered as an aqueous extract, can activate immunity to suppress tumor and is more effective than each drug on its own in vitro and in vivo. Based on network pharmacology analysis, PIK3R1 is the core target for the anti-tumor immunity activity of combined Huangqi and Danggui. Molecular docking analysis shows 6 components of the combined Danggui and Huangqi extract (quercetin, jaranol, isorhamnetin, kaempferol, calycosin, and suchilactone) that bind to PIK3R1. Jaranol is the most important component against breast cancer. The suchilactone/jaranol combination and, especially, the suchilactone/kaempferol combination are key for immunity enhancement and the anti-tumor effects of the extract.

Conclusions: The combination of Huangqi and Danggui can activate immunity to suppress breast cancer and is more effective than the individual drugs alone.

Keywords: Danggui; Huangqi; anti‐tumor; immunity.

FIGURE 1

The compatibility of supplement‐Qi TCMs.

FIGURE 2

The combination of Danggui and Huangqi has anti‐tumor effects and improves immunity. (A) Effects of different proportions of Danggui and Huangqi on the activity of murine spleen cells and 4T1 cells. (B) The effects of the Danggui and Huangqi combination (1:3) on CD4, CD8, and IFN‐γ expression in co‐culture cells were evaluated using high content screening. (C) The level of IFN‐γ was tested in co‐culture supernatant using ELISA after treatment with combined Danggui and Huangqi (1:3). (D–F) Effects of different proportions of Danggui and Huangqi on the expression of CD4 (D), CD8/CD4 (E), and IFN‐γ (F) in co‐cultured cells. Data shown are the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with the control group.

FIGURE 3

Combined Danggui and Huangqi inhibit breast cancer growth. (A) The therapeutic effects of combined Danggui and Huangqi (1:3) were examined for 4T1 cells xenograft mice. (B) Tumor weight and growth curves were tested after treatment of different doses of combined Danggui and Huangqi (1:3). (C) Tumor index and volume were tested after treatment with a high dose of combined Danggui and Huangqi (1:3) or single drugs. Data shown are the mean ± SEM of three independent experiments. *p < 0.05, and **p < 0.01, compared with the model group.

FIGURE 4

Combined Danggui and Huangqi may inhibit breast cancer growth through immune activation. (A–F) The index of heart (A), liver (B), lung (C), kidney (D), thymus (E), and spleen (F) were tested after treatment with different doses of combined Danggui and Huangqi (1:3) or single drugs. Data shown are the mean ± SEM of three independent experiments. (G) H&E staining of heart, liver, spleen, lung, and kidney after a high dose of combined Danggui and Huangqi. *p < 0.05, and **p < 0.01, compared with the model group.

FIGURE 5

Combined Danggui and Huangqi activate immunity. (A) Flow cytometric analysis of CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, and CD3⁺IFN‐γ T cells. (B) Flow cytometric analysis of CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, and CD3⁺IFN‐γ T cells tested after treatment with a high dose of combined Danggui and Huangqi (1:3) or single drugs. (C) Flow cytometric analysis of CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, and CD3⁺IFN‐γ T cells tested after treatment with a different dose of combined Danggui and Huangqi (1:3). (D) Flow cytometric analysis of CD45⁺B220⁺ cells, NK‐1.1 cells, CD11c⁺MHCI⁺ cells, CD11c⁺MHCII⁺ cells, and CD11b⁺CD11c⁺CD80⁺ cells tested after treatment with a high dose of combined Danggui and Huangqi (1:3) or single drugs. (E) Flow cytometric analysis of CD45⁺B220⁺ cells, NK‐1.1 cells, CD11c⁺MHCI⁺ cells, CD11c⁺MHCII⁺ cells, and CD11b⁺CD11c⁺CD80⁺ cells tested after treatment with different doses of combined Danggui and Huangqi (1:3). Data shown are the mean ± SEM of three independent experiments. *p < 0.05, and **p < 0.01, compared with the model group.

FIGURE 6

Network pharmacological analysis of combined Danggui and Huangqi. (A) Network analysis of components and gene targets from combined Danggui and Huangqi. (B) The PPI network was established using STRING and Cytoscape. (C) The KEGG pathway enrichment analysis. (D) Biological processes, cellular components, and molecular functions of targeted genes. (E) Cytoscape was used to analyze the network among TCMs, active ingredients, protein targets, the KEGG pathways, and tumor immunity.

FIGURE 7

PIK3R1 may be a core hub targets for anti‐tumor immunity of combined Danggui and Huangqi. (A) Molecular docking of PIK3R1 with 6 ingredients (quercetin, jaranol, isorhamnetin, kaempferol, calycosin, and suchilactone). The score indicates the binding energy. (B) The mRNA expression of PIK3R1 was tested in 4T1 cells treated with quercetin, jaranol, isorhamnetin, kaempferol, calycosin, or suchilactone. Data shown are the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001, compared with the control group.

FIGURE 8

Jaranol is the most important component in the combined Danggui and Huangqi to enhance immunity and inhibit tumor by targeting PIK3R1. (A) Effects of 6 ingredients on the activity of murine spleen cells and 4T1 cells. (B) The level of IFN‐γ was tested in co‐culture supernatant using ELISA after treatment with 6 ingredients. (C) The effects of 6 ingredients on CD4, CD8, and IFN‐γ expression in co‐culture cells were evaluated using high content screening. (D) Effects of 6 ingredients on the expression of CD4, CD8/CD4, and IFN‐γ in co‐culture cells. (E) Effects of jaranol on the activity of murine spleen cells and 4T1 cells were tested after the pretreatment of LY294002. (F) Effects of jaranol on the expression of CD4, CD8/CD4, and IFN‐γ in co‐cultured cells were tested after the pretreatment of LY294002. Data shown are the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with the control group.

FIGURE 9

The combination of suchilactone and kaempferol is key for anti‐tumor effect of combined Danggui and Huangqi. (A) Effects of combined suchilactone and the five Huangqi‐derived ingredients (suchilactone/quercetin, suchilactone/kaempferol, suchilactone/calycosin, suchilactone/jaranol, and suchilactone/isorhamnetin) on the activity of murine spleen cells and 4T1 cells. (B) The level of IFN‐γ was tested in co‐culture supernatant using ELISA after the treatment with combined suchilactone and the five Huangqi‐derived ingredients. (C) The effects of combined suchilactone and the five Huangqi‐derived ingredients on CD4, CD8, and IFN‐γ expression in co‐culture cells were evaluated using high content screening. (D) Effects of combined suchilactone and the five Huangqi‐derived ingredients on the expression of CD4, CD8/CD4, and IFN‐γ in co‐cultured cells. (E) Effects of combined suchilactone and the five Huangqi‐derived ingredients on the activity of murine spleen cells and 4T1 cells were tested after the pretreatment of LY294002. (F) Effects of the suchilactone/kaempferol combination on the expression of CD4, CD8/CD4, and IFN‐γ in co‐cultured cells were tested after the pretreatment of LY294002. Data shown are the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with the control group.

FIGURE 10

The anti‐tumor effect of combined suchilactone and kaempferol is stronger than the suchilactone/kaempferol/jaranol combination. (A and B) Effects of suchilactone/kaempferol combination, suchilactone/jaranol combination, and suchilactone/kaempferol/jaranol combination on the activity of murine spleen cells (A) and 4T1 cells (B). (C) High content screening detected the expression of CD4, CD8, and IFN‐γ in co‐cultured cells treated by suchilactone/kaempferol combination, suchilactone/jaranol combination, and suchilactone/kaempferol/jaranol combination. (D) Effects of suchilactone/kaempferol combination, suchilactone/jaranol combination, and suchilactone/kaempferol/jaranol combination on the expression of CD4, CD8/CD4, and IFN‐γ in co‐cultured cells. Data shown are the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with the control group.